Peptide probe for rapid and specific detection of amyloid aggregation

ABSTRACT

A method for use of a peptide probe that generates fluorescence signals rapidly upon recognition of various Aβ aggregates without significant perturbation of samples. The present peptide probes display an increase in fluorescence signals upon coincubation with Aβ oligomers, but neither monomeric/dimeric species nor fibrils. The detection can occur within an hour or two without any additional sample preparation and incubation steps.

STATEMENT OF RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/856,209 having a filing date of 13 Aug. 2012, which is based on andclaims the benefit of U.S. Provisional Patent Application No. 61/234,083having a filing date of 14 Aug. 2010, currently pending, both of whichare incorporated herein in their entireties by this reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on 16 Sep. 2011, isnamed 484673US.txt and is 2,831 bytes in size.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention is generally related to the field of peptide probes, tothe field of peptide probes for the detection of amyloid aggregation,and to the field of peptide probes for the rapid and specific detectionof amyloid aggregation.

2. Prior Art

Aggregation of a 39-43 amino acid peptide, beta amyloid (Aβ) (Kang, J.,et al., The precursor of Alzheimer's disease amyloid A4 proteinresembles a cell-surface receptor, Nature 325, 733-736, 1987; Roher, A.E., et al., beta-Amyloid-(1-42) is a major component of cerebrovascularamyloid deposits: implications for the pathology of Alzheimer disease,Proc Natl Acad Sci USA 90, 10836-10840, 1993), into a fibril viaformation of nuclei (Kusumoto, Y., et al., Temperature dependence ofamyloid beta-protein fibrillization, Proc Natl Acad Sci USA 95,12277-12282, 1998; Teplow, D. B., et al., Elucidating amyloidbeta-protein folding and assembly: A multidisciplinary approach, AccChem Res 39, 635-645, 2006; Fernandez-Busquets, X., et al., Recentstructural and computational insights into conformational diseases, CurrMed Chem 15, 1336-1349, 2008; Lazo, N. D., et al., On the nucleation ofamyloid beta-protein monomer folding, Protein Sci 14, 1581-1596, 2005;Wetzel, R., et al., Plasticity of amyloid fibrils. Biochemistry 46,1-10, 2007) is believed to be implicated in the pathology of Alzheimer'sdisease (AD), which is a neurodegenerative disorder characterized by aprogressive loss of cognitive functions and by neuropathologicalfeatures comprising amyloid deposits and neuronal losses in the brain(Hardy, J., et al., The amyloid hypothesis of Alzheimer's disease:progress and problems on the road to therapeutics, Science 297, 353-356,2002; Mattson, M. P., Pathways towards and away from Alzheimer'sdisease, Nature 430, 631-639, 2004; Haass, C., et al., Soluble proteinoligomers in neurodegeneration: lessons from the Alzheimer's amyloidbeta-peptide, Nat Rev Mol Cell Biol 8, 101-112, 2007).

Low molecular weight Aβ species, such as monomers and dimmers, are nottoxic (Haass, C., et al., (2007) Soluble protein oligomers inneurodegeneration: lessons from the Alzheimer's amyloid beta-peptide,Nat Rev Mol Cell Biol 8, 101-112, 2007; Kayed, R., et al., Commonstructure of soluble amyloid oligomers implies common mechanism ofpathogenesis, Science 300, 486-489, 2003; Kayed, R., et al.,Permeabilization of lipid bilayers is a common conformation-dependentactivity of soluble amyloid oligomers in protein misfolding diseases, JBiol Chem 279, 46363-46366, 2004; Klyubin, I., et al., Amyloid betaprotein immunotherapy neutralizes Abeta oligomers that disrupt synapticplasticity in vivo, Nat Med 11, 556-561, 2005). A considerable amount ofdata has identified the soluble Aβ oligomers as potentially significanttoxic agents (Haass, C., et al., Soluble protein oligomers inneurodegeneration: lessons from the Alzheimer's amyloid beta-peptide,Nat Rev Mol Cell Biol 8, 101-112, 2007; Glabe, C. G., Common mechanismsof amyloid oligomer pathogenesis in degenerative disease, NeurobiolAging 27, 570-575, 2006). However, the possibility that the Aβ fibrilsmay be associated with neurotoxicity cannot be ruled out, sincefibrillar aggregates can serve as a pool of soluble intermediate speciesthrough a dynamic exchange with monomers or oligomers (Id.; O'Nuallain,B., et al., Thermodynamics of A beta(1-40) amyloid fibril elongation,Biochemistry 44, 12709-12718, 2005; Martins, I. C., et al., Lipidsrevert inert Abeta amyloid fibrils to neurotoxic protofibrils thataffect learning in mice, EMBO J 27, 224-233, 2008). Toxic oligomers arekinetic intermediates, and can display changes in conformation and toxiceffects by subtle environmental changes (Teplow, D. B., et al.,Elucidating amyloid beta-protein folding and assembly: Amultidisciplinary approach, Acc Chem Res 39, 635-645, 2006; Wetzel, R.,et al., Plasticity of amyloid fibrils, Biochemistry 46, 1-10, 2007;Haass, C., et al., Soluble protein oligomers in neurodegeneration:lessons from the Alzheimer's amyloid beta-peptide, Nat Rev Mol Cell Biol8, 101-112, 2007; Klyubin, I., et al., Amyloid beta proteinimmunotherapy neutralizes Abeta oligomers that disrupt synapticplasticity in vivo, Nat Med 11, 556-561, 2005).

Determination of population profiles of different aggregate species isstrongly required to understand the molecular causes of Aβ aggregationas well as toxic processes in AD. However, the complex nature of Aβaggregation, including the generation of transient aggregateintermediates, impedes the establishment of a functional correlationbetween Aβ aggregation characteristics and their cellular/clinicalmanifestations. A quantitative measurement of aggregate species must bedone rapidly without significant perturbation of samples for high levelaccuracy, as aggregates including toxic soluble oligomers are likely toundergo further structural changes during the additional samplepreparation and incubation steps (Haass, C., et al., Soluble proteinoligomers in neurodegeneration: lessons from the Alzheimer's amyloidbeta-peptide, Nat Rev Mol Cell Biol 8, 101-112, 2007; Chromy, B. A., etal., Self-assembly of Abeta(1-42) into globular neurotoxins.Biochemistry 42, 12749-12760, 2003; Hoyer, W., et al., Dependence ofalpha-synuclein aggregate morphology on solution conditions, J Mol Biol322, 383-393, 2002). Rapid and specific detection of distinctamyloidogenic species is therefore quintessential for the establishmentof a reliable correlation between aggregation profiles and theircellular/clinical manifestations as well as achieving betterunderstanding of the determinants of aggregation.

Inaccurate quantification of various aggregate species would result inthe gap seen between basic scientific discovery and cellular/clinicalmanifestations, and the discrepancy among observations from animal modelstudies. The currently available compounds or methods, however, eitherdo not distinguish different aggregate species or are inappropriate forrapid, non-perturbative detection due to the requirement of additionalsample preparation and incubation steps (Kayed, R., et al., Commonstructure of soluble amyloid oligomers implies common mechanism ofpathogenesis, Science 300, 486-489, 2003; Williams, A. D., et al.,Structural properties of Abeta protofibrils stabilized by a smallmolecule, Proc Natl Acad Sci USA 102, 7115-7120, 2005; Kayed, R., etal., Conformation-dependent anti-amyloid oligomer antibodies, MethodsEnzymol 413, 326-344, 2006; Kayed, R., et al., Fibril specific,conformation dependent antibodies recognize a generic epitope common toamyloid fibrils and fibrillar oligomers that is absent in prefibrillaroligomers, Mol Neurodegener 2, 18, 2007; Linke, R. P., et al.,High-sensitivity diagnosis of AA amyloidosis using Congo red andimmunohistochemistry detects missed amyloid deposits, J HistochemCytochem 43, 863-869, 1995; LeVine, H., 3^(rd), Quantification ofbeta-sheet amyloid fibril structures with thioflavin T, Methods Enzymol309, 274-284, 1999).

Accordingly, there is always a need for improved probes for thedetection of amyloid aggregation. There also always is a need forimproved peptide probes for the rapid and specific detection of amyloidaggregation. It is to these needs, among others, that this invention isdirected.

BRIEF SUMMARY OF THE INVENTION

Determination of population profiles of different aggregate species isstrongly required to understand the molecular causes of beta-amyloid(Aβ) aggregation as well as toxic processes in Alzheimer's disease (AD).A quantitative measurement of aggregate species must be done rapidlywithout perturbation of samples for high level accuracy, as aggregatesincluding toxic soluble oligomers are likely to undergo furtherstructural changes during the additional sample preparation andincubation steps. The present invention is a design of a peptide probethat may generate fluorescence signals rapidly upon recognition ofvarious Aβ aggregates without significant perturbation of samples. Thepresent peptide probe displays an increase in fluorescence signals uponcoincubation with Aβ oligomers, but neither monomeric/dimeric speciesnor fibrils. The detection can occur within an hour or two without anyadditional sample preparation and incubation steps.

The peptide probe can be used for detection of toxic Aβ oligomers fordiagnostic applications of Alzheimer's disease on tissue samples orbiological fluids, for screening of therapeutic agents that can alterthe protein aggregation process and the resulting aggregate toxicity,and provides a sensitive and specific assay for Aβ aggregate formationin biochemical studies.

These uses and features, and other uses, features and advantages of thepresent invention, will become more apparent to those of ordinary skillin the relevant art when the following detailed description of thepreferred embodiments is read in conjunction with the appended figures.

BRIEF SUMMARY OF THE FIGURES

FIG. 1A is the amino acid sequences of Aβ40, Aβ42 and PG46. The AβN-terminal domain is shown in plain letters. The Aβ HCD domain is doubleunderlined. The Aβ C-terminal domain is single underlined. The linkerregion in Aβ40 and Aβ42, and the signal domain in PG46 are shown in bolditalic letters.

FIG. 1B is the proposed mechanism of modulation of FlAsH fluorescence ofPG46 through association with Aβ oligomers.

FIG. 1C is the FlAsH-concentration dependent FlAsH fluorescence of PG46.The concentration of PG46 was 0.05 mg/ml. The excitation wavelength was508 nm.

FIG. 2A is the thioflavin T fluorescence of Aβ40 monomers/dimers (Aβ40M/D), Aβ42 monomers/dimers (Aβ42 M/D), Aβ40 oligomers (Aβ40 O), Aβ42oligomers (Aβ42 O), Aβ40 fibrils (Aβ42 F), and Aβ42 fibrils (Aβ42 F).Samples were prepared according to (left) the DMSO or HFIP protocol and(right) the urea protocol. Thioflavin T fluorescence of Aβ at 0.25 mg/mlincubated in PBSA containing 0.4M urea after 250 hr incubation at 37° C.with constant shaking at 250 rpm was shown for comparison.

FIG. 2B are TEM images of Aβ40 M/D, Aβ42 M/D, Aβ40 O, Aβ42 O, Aβ42 F andAβ42 F. All samples were prepared according to the DMSO or HFIPprotocol. Scale bars represent 200 nm.

FIG. 2C is a circular dichroism (CD) spectra of Aβ40 O and Aβ40 M/Dprepared according to the urea protocol.

FIG. 3 are FlAsH fluorescence spectra of PG46 when mixed with samplescontaining (A and G) Aβ40 monomers/dimers, (B, H and I) Aβ40 oligomers,(C) Aβ40 fibrils, (D) Aβ42 monomers/dimers, (E) Aβ42 oligomers and (F)Aβ42 fibrils. Aβ samples were prepared according to (A, B, D, E and I)the DMSO, (C and F) HFIP and (G and H) urea protocols. The massconcentrations of Aβ were 0.05 mg/ml in (I) and 0.1 mg/ml in all theother samples, respectively. Fluorescence spectra of samples containingAβ only, PG46 only, and Aβ+PG46 are represented by black long dashlines, green solid lines, and red dot lines, respectively. For FlAsHmeasurements, samples of Aβ only, PG46 only and Aβ+PG46 were incubatedat 25° C. without agitation for 1 hr prior to addition of FlAsH followedby additional 1 hr incubation before fluorescence measurements. Theexcitation wavelength was 508 nm. The mass concentration of PG46 was0.05 mg/ml. n≧3. The error bar represents one standard deviation.

FIG. 4 is a representative size exclusion chromatography (SEC) elutionprofile of Aβ40 at 0.6 mg/ml in the aqueous buffer. Mobile phase was thesame aqueous buffer. The mobile phase flow rate was 0.1 ml/min andelution peaks were detected by UV absorbance at 280 nm. Molecular massof Aβ40 peak was determined by calibration of column using insulin chainB (3.5 kDa), ubiquitin (8.5 kDa), ribonuclease A (13.7 kDa) and bovineserum albumin (67 kDa). The X marks above the SEC spectrum representelution times of bovine serum albumin, ribonuclease A, ubiquitin andinsulin chain B from the left to the right, respectively. The fractionof Aβ40 solution eluting at 16 min (corresponding to the largest peak)was collected and named “Aβ40 monomer/dimer” samples. “Aβ42monomer/dimer” samples were prepared similarly.

FIG. 5A is a circular dichroism (CD) spectra of 0.5 mg/ml PG46 duringincubation in HFIP at RT. CD signals were changed over time during thefirst several minute of incubation of PG46 in HFIP, then remained sameafter 1 hr, implying neither further occurrence of dissolution norα-helical formation of PG46.

FIG. 5B is a dot blot assay of PG46 using an oligomer-specificpolyclonal antibody, A11.

FIG. 6A is a FlAsH fluorescence of PG46 when mixed with Aβ40 Dutcholigomer samples. Fluorescence spectra of samples containing Aβ only,PG46 only, and Aβ+PG46 are represented by black long dash lines, greensolid lines and red dot lines, respectively. For FlAsH measurements,samples of Aβ only, PG46 only and Aβ+PG46 were incubated at 25° C.without agitation for 1 hr prior to addition of FlAsH followed byadditional 1 hr incubation. The excitation wavelength was 508 nm. Themass concentration of PG46 was 0.05 mg/ml. n≧3. The error bar representsone standard deviation.

FIG. 6B is a FlAsH fluorescence of PG46 when mixed with Aβ40 Articoligomer samples. Fluorescence spectra of samples containing Aβ only,PG46 only, and Aβ+PG46 are represented by black long dash lines, greensolid lines and red dot lines, respectively. For FlAsH measurements,samples of Aβ only, PG46 only and Aβ+PG46 were incubated at 25° C.without agitation for 1 hr prior to addition of FlAsH followed byadditional 1 hr incubation. The excitation wavelength was 508 nm. Themass concentration of PG46 was 0.05 mg/ml. n≧3. The error bar representsone standard deviation.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Alzheimer's disease (AD) is a neurodegenerative disorder characterizedby a progressive loss of cognitive functions and by neuropathologicalfeatures comprising amyloid deposits and neuronal losses in brain(Hardy, J., et al., The amyloid hypothesis of Alzheimer's disease:progress and problems on the road to therapeutics, Science 297, 353-356,2002; Mattson, M. P., Pathways towards and away from Alzheimer'sdisease, Nature 430, 631-639 (2004). The principal constituent ofamyloid deposits is a 40-42 amino acid peptide, referred to as β amyloid(Aβ) (FIG. 1A) (Kang, J., et al., The precursor of Alzheimer's diseaseamyloid A4 protein resembles a cell-surface receptor, Nature 325,733-736, 1987; Roher, A. E., et al., beta-Amyloid-(1-42) is a majorcomponent of cerebrovascular amyloid deposits: implications for thepathology of Alzheimer disease, Proc Natl Acad Sci USA 90, 10836-10840,1993). Aβ is derived from the amyloid precursor protein (APP) byproteolytic cleavage¹ (Kang, J., et al., The precursor of Alzheimer'sdisease amyloid A4 protein resembles a cell-surface receptor, Nature325, 733-736, 1987). Aβ contains the hydrophilic N-terminus (D1-K16),the hydrophobic central domain (HCD, L17-A21), the linker region(E22-A30), and the hydrophobic C-terminus (I31-V40 or I31-A42 in Aβ40and Aβ42, respectively) (FIG. 1A) (Id.). Whereas the N-terminus is notessential in aggregation (Pike, C. J., et al., Amino-terminal deletionsenhance aggregation of beta-amyloid peptides in vitro, J Biol Chem 270,23895-23898, 1995), HCD and the C-terminus of Aβ have been found to becritical in Aβ self-assembly and aggregation-prone (Tjernberg, L. O., etal., Arrest of beta-amyloid fibril formation by a pentapeptide ligand, JBiol Chem 271, 8545-8548, 1996; Soto, C., et al., Beta-sheet breakerpeptides inhibit fibrillogenesis in a rat brain model of amyloidosis:implications for Alzheimer's therapy, Nat Med 4, 822-826, 1998; Liu, R.,et al., Residues 17-20 and 30-35 of beta-amyloid play critical roles inaggregation, J Neurosci Res 75, 162-171, 2004; Christopeit, T., et al.,Mutagenic analysis of the nucleation propensity of oxidized Alzheimer'sbeta-amyloid peptide, Protein Sci 14, 2125-2131, 2005). Aggregation ofAβ into fibrils via formation of nuclei is believed to be implicated inthe pathology of AD (Kusumoto, Y., et al., Temperature dependence ofamyloid beta-protein fibrillization, Proc Natl Acad Sci USA 95,12277-12282, 1998; Teplow, D. B., et al., Elucidating amyloidbeta-protein folding and assembly: A multidisciplinary approach, AccChem Res 39, 635-645, 2006; Fernandez-Busquets, X., et al., Recentstructural and computational insights into conformational diseases, CurrMed Chem 15, 1336-1349, 2008; Lazo, N. D., et al., On the nucleation ofamyloid beta-protein monomer folding, Protein Sci 14, 1581-1596, 2005;Wetzel, R., et al., Plasticity of amyloid fibrils, Biochemistry 46,1-10, 2007; Hardy, J., et al., The amyloid hypothesis of Alzheimer'sdisease: progress and problems on the road to therapeutics, Science 297,353-356, 2002; Mattson, M. P., Pathways towards and away fromAlzheimer's disease, Nature 430, 631-639, 2004; Haass, C. et al.,Soluble protein oligomers in neurodegeneration: lessons from theAlzheimer's amyloid beta-peptide, Nat Rev Mol Cell Biol 8, 101-112,2007).

Experimental and simulation studies have suggested that conformationalrearrangement of Aβ occurs during the assembly of monomers intooligomers, then into fibrils (Teplow, D. B., et al., Elucidating amyloidbeta-protein folding and assembly: A multidisciplinary approach, AccChem Res 39, 635-645, 2006; Fernandez-Busquets, X., et al., Recentstructural and computational insights into conformational diseases, CurrMed Chem 15, 1336-1349, 2008; Lazo, N. D., et al., On the nucleation ofamyloid beta-protein monomer folding, Protein Sci 14, 1581-1596, 2005;Wetzel, R., et al., Plasticity of amyloid fibrils, Biochemistry 46,1-10, 2007). Monomeric Aβ is, in large, irregularly structured (Zhang,S., et al., The Alzheimer's peptide a beta adopts a collapsed coilstructure in water, J Struct Biol 130, 130-141, 2000; Lee, J. P., etal., 1H NMR of A beta amyloid peptide congeners in water solution,Conformational changes correlate with plaque competence, Biochemistry34, 5191-5200, 1995; Riek, R., et al., NMR studies in aqueous solutionfail to identify significant conformational differences between themonomeric forms of two Alzheimer peptides with widely differentplaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox), Eur J Biochem268, 5930-5936, 2001; Hou, L., et al., Solution NMR studies of the Abeta(1-40) and A beta(1-42) peptides establish that the Met35 oxidationstate affects the mechanism of amyloid formation, J Am Chem Soc 126,1992-2005, 2004). Various oligomeric species of Aβ were observed duringits aggregation from monomeric states in vivo and in vitro (Klyubin, I.,et al., Amyloid beta protein immunotherapy neutralizes Abeta oligomersthat disrupt synaptic plasticity in vivo, Nat Med 11, 556-561, 2005;Goldsbury, C., et al., Multiple assembly pathways underlie amyloid-betafibril polymorphisms, J Mol Biol 352, 282-298, 2005; Harper, J. D., etal., Observation of metastable Abeta amyloid protofibrils by atomicforce microscopy, Chem Biol 4, 119-125, 1997; Lansbury, P. T., Jr.,Evolution of amyloid: what normal protein folding may tell us aboutfibrillogenesis and disease, Proc Natl Acad Sci USA 96, 3342-3344, 1999;Walsh, D. M., et al., Amyloid beta-protein fibrillogenesis. Structureand biological activity of protofibrillar intermediates, J Biol Chem274, 25945-25952, 1999; Huang, T. H., et al., Structural studies ofsoluble oligomers of the Alzheimer beta-amyloid peptide, J Mol Biol 297,73-87, 2000; Nichols, M. R., et al., Growth of beta-amyloid(1-40)protofibrils by monomer elongation and lateral association.Characterization of distinct products by light scattering and atomicforce microscopy, Biochemistry 41, 6115-6127, 2002; Yong, W., et al.,Structure determination of micelle-like intermediates in amyloidbeta-protein fibril assembly by using small angle neutron scattering,Proc Natl Acad Sci USA 99, 150-154, 2002; Hoshi, M., et al., Sphericalaggregates of beta-amyloid (amylospheroid) show high neurotoxicity andactivate tau protein kinase I/glycogen synthase kinase-3beta, Proc NatlAcad Sci USA 100, 6370-6375, 2003; Barghorn, S., et al., Globularamyloid beta-peptide oligomer—a homogenous and stable neuropathologicalprotein in Alzheimer's disease, J Neurochem 95, 834-847, 2005; Chimon,S., et al., Capturing intermediate structures of Alzheimer'sbeta-amyloid, Abeta(1-40), by solid-state NMR spectroscopy, J Am ChemSoc 127, 13472-13473, 2005; Chimon, S., et al., Evidence of fibril-likebeta-sheet structures in a neurotoxic amyloid intermediate ofAlzheimer's beta-amyloid, Nat Struct Mol Biol, 2007; Hepler, R. W., etal., Solution state characterization of amyloid beta-derived diffusibleligands, Biochemistry 45, 15157-15167, 2006; Losic, D., et al., Highresolution scanning tunnelling microscopy of the beta-amyloid protein(Abeta1-40) of Alzheimer's disease suggests a novel mechanism ofoligomer assembly, J Struct Biol 155, 104-110, 2006; Mastrangelo, I. A.,et al., High-resolution atomic force microscopy of soluble Abeta42oligomers, J Mol Biol 358, 106-119, 2006; Walsh, D. M., et al.,Naturally secreted oligomers of amyloid beta protein potently inhibithippocampal long-term potentiation in vivo, Nature 416, 535-539, 2002;Podlisny, M. B., et al., Aggregation of secreted amyloid beta-proteininto sodium dodecyl sulfate-stable oligomers in cell culture, J BiolChem 270, 9564-9570, 1995; Walsh, D. M., et al., The oligomerization ofamyloid beta-protein begins intracellularly in cells derived from humanbrain, Biochemistry 39, 10831-10839, 2000; Lesne, S., et al., A specificamyloid-beta protein assembly in the brain impairs memory, Nature 440,352-357, 2006). Several Aβ oligomers, such as spherical andprotofibrillar species, were proposed as the structural units from whichlarger aggregates emerge (Goldsbury, C., et al., Multiple assemblypathways underlie amyloid-beta fibril polymorphisms, J Mol Biol 352,282-298, 2005; Harper, J. D., et al., Observation of metastable Abetaamyloid protofibrils by atomic force microscopy, Chem Biol 4, 119-125,1997; Lansbury, P. T., Jr., Evolution of amyloid: what normal proteinfolding may tell us about fibrillogenesis and disease, Proc Natl AcadSci USA 96, 3342-3344, 1999; Walsh, D. M., et al., Amyloid beta-proteinfibrillogenesis. Structure and biological activity of protofibrillarintermediates, J Biol Chem 274, 25945-25952, 1999; Huang, T. H., et al.,Structural studies of soluble oligomers of the Alzheimer beta-amyloidpeptide, J Mol Biol 297, 73-87, 2000; Nichols, M. R., et al., Growth ofbeta-amyloid(1-40) protofibrils by monomer elongation and lateralassociation. Characterization of distinct products by light scatteringand atomic force microscopy, Biochemistry 41, 6115-6127, 2002; Yong, W.,et al., Structure determination of micelle-like intermediates in amyloidbeta-protein fibril assembly by using small angle neutron scattering,Proc Natl Acad Sci USA 99, 150-154, 2002; Hoshi, M., et al., Sphericalaggregates of beta-amyloid (amylospheroid) show high neurotoxicity andactivate tau protein kinase I/glycogen synthase kinase-3beta, Proc NatlAcad Sci USA 100, 6370-6375, 2003; Barghorn, S., et al., Globularamyloid beta-peptide oligomer—a homogenous and stable neuropathologicalprotein in Alzheimer's disease, J Neurochem 95, 834-847, 2005; Chimon,S., et al., Capturing intermediate structures of Alzheimer'sbeta-amyloid, Abeta(1-40), by solid-state NMR spectroscopy, J Am ChemSoc 127, 13472-13473, 2005; Chimon, S., et al., Evidence of fibril-likebeta-sheet structures in a neurotoxic amyloid intermediate ofAlzheimer's beta-amyloid, Nat Struct Mol Biol, 2007; Hepler, R. W., etal., Solution state characterization of amyloid beta-derived diffusibleligands, Biochemistry 45, 15157-15167, 2006; Losic, D., et al., Highresolution scanning tunnelling microscopy of the beta-amyloid protein(Abeta1-40) of Alzheimer's disease suggests a novel mechanism ofoligomer assembly, J Struct Biol 155, 104-110, 2006; Mastrangelo, I. A.,et al., High-resolution atomic force microscopy of soluble Abeta42oligomers. J Mol Biol 358, 106-119, 2006). These oligomers displayedsubstantial β strand structures (Lansbury, P. T., Jr., Evolution ofamyloid: what normal protein folding may tell us about fibrillogenesisand disease, Proc Natl Acad Sci USA 96, 3342-3344, 1999; Walsh, D. M.,et al., Amyloid beta-protein fibrillogenesis. Structure and biologicalactivity of protofibrillar intermediates, J Biol Chem 274, 25945-25952,1999; Huang, T. H., et al., Structural studies of soluble oligomers ofthe Alzheimer beta-amyloid peptide, J Mol Biol 297, 73-87, 2000;Nichols, M. R., et al., Growth of beta-amyloid(1-40) protofibrils bymonomer elongation and lateral association, Characterization of distinctproducts by light scattering and atomic force microscopy, Biochemistry41, 6115-6127, 2002; Barghorn, S., et al., Globular amyloid beta-peptideoligomer—a homogenous and stable neuropathological protein inAlzheimer's disease, J Neurochem 95, 834-847, 2005; Chimon, S., et al.,Capturing intermediate structures of Alzheimer's beta-amyloid,Abeta(1-40), by solid-state NMR spectroscopy, J Am Chem Soc 127,13472-13473, 2005; Chimon, S., et al., Evidence of fibril-likebeta-sheet structures in a neurotoxic amyloid intermediate ofAlzheimer's beta-amyloid, Nat Struct Mol Biol, 2007; Losic, D., et al.,High resolution scanning tunnelling microscopy of the beta-amyloidprotein (Abeta1-40) of Alzheimer's disease suggests a novel mechanism ofoligomer assembly, J Struct Biol 155, 104-110, 2006; Mastrangelo, I. A.,et al., High-resolution atomic force microscopy of soluble Abeta42oligomers. J Mol Biol 358, 106-119, 2006). Protofibrils of Aβ,curvilinear structures which appeared as strings of the sphericalparticles in atomic force microscopy (AFM) images, can further grow intofibrils by association with monomers or other protofibrils (Lansbury, P.T., Jr., Evolution of amyloid: what normal protein folding may tell usabout fibrillogenesis and disease, Proc Natl Acad Sci USA 96, 3342-3344,1999; Walsh, D. M., et al., Amyloid beta-protein fibrillogenesis.Structure and biological activity of protofibrillar intermediates, JBiol Chem 274, 25945-25952, 1999; Huang, T. H., et al., Structuralstudies of soluble oligomers of the Alzheimer beta-amyloid peptide, JMol Biol 297, 73-87, 2000; Nichols, M. R., et al., Growth ofbeta-amyloid(1-40) protofibrils by monomer elongation and lateralassociation, Characterization of distinct products by light scatteringand atomic force microscopy, Biochemistry 41, 6115-6127, 2002; Losic,D., et al., High resolution scanning tunnelling microscopy of thebeta-amyloid protein (Abeta1-40) of Alzheimer's disease suggests a novelmechanism of oligomer assembly, J Struct Biol 155, 104-110, 2006;Mastrangelo, I. A., et al., High-resolution atomic force microscopy ofsoluble Abeta42 oligomers. J Mol Biol 358, 106-119, 2006). Solubleoligomers may form β strand-turn-β strand or β strand-loop-β strandstructures where the turn or loop may be found within the linker region(Barghorn, S., et al., Globular amyloid beta-peptide oligomer—ahomogenous and stable neuropathological protein in Alzheimer's disease,J Neurochem 95, 834-847, 2005; Chimon, S., et al., Evidence offibril-like beta-sheet structures in a neurotoxic amyloid intermediateof Alzheimer's beta-amyloid, Nat Struct Mol Biol, 2007; Losic, D., etal., High resolution scanning tunnelling microscopy of the beta-amyloidprotein (Abeta1-40) of Alzheimer's disease suggests a novel mechanism ofoligomer assembly, J Struct Biol 155, 104-110, 2006; Lazo, N. D., etal., On the nucleation of amyloid beta-protein monomer folding, ProteinSci 14, 1581-1596, 2005; Hoyer, W., et al., Stabilization of abeta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibitsamyloid formation. Proc Natl Acad Sci USA 105, 5099-5104, 2008; Habicht,G., et al., Directed selection of a conformational antibody domain thatprevents mature amyloid fibril formation by stabilizing Abetaprotofibrils. Proc Natl Acad Sci USA 104, 19232-19237, 2007). Amyloidfibrils contain in-register cross β sheets running perpendicular to thelong axis of fibrils. Solid state NMR has been successfully used fordetermination of Aβ fibril structures (Petkova, A. T., et al., Astructural model for Alzheimer's beta-amyloid fibrils based onexperimental constraints from solid state NMR, Proc Natl Acad Sci USA99, 16742-16747, 2002; Luhrs, T., et al., 3D structure of Alzheimer'samyloid-beta(1-42) fibrils, Proc Natl Acad Sci USA 102, 17342-17347,2005).

In an Aβ40 fibril model structure, residues D1-E11 of Aβ40 arestructurally disordered while the rest of the sequence forms a βstrand-loop-β strand motif. β strands are populated within residuesV12-V24 and A30-V40, and held together by intermolecular hydrogenbonding parallel to the fibril long axis. A loop structure is formedwithin residues G25-G29. Consistent with this model, hydrogen-deuterium(HD) exchange studies of Aβ40 fibrils indicated the presence ofprotected core structures within K16-V36 with rapidly exchangeable amideprotons at G25 and S26 (Whittemore, N. A., et al., Hydrogen-deuterium(H/D) exchange mapping of Abeta 1-40 amyloid fibril secondary structureusing nuclear magnetic resonance spectroscopy, Biochemistry 44,4434-4441, 2005). The formation of this loop is mediated by cross-strandside chain interactions and a salt bridge between D23 and K28 (Petkova,A. T., et al., A structural model for Alzheimer's beta-amyloid fibrilsbased on experimental constraints from solid state NMR, Proc Natl AcadSci USA 99, 16742-16747, 2002). This fibril model is largely consistentwith Aβ40 fibril structures suggested from alanine and proline scanningmutagenesis analyses, except for the presence of two turns located atE22-D23 and G29-A30 (Williams, A. D., et al., Alanine scanningmutagenesis of Abeta(1-40) amyloid fibril stability, J Mol Biol 357,1283-1294, 2006; Williams, A. D., et al., Mapping abeta amyloid fibrilsecondary structure using scanning proline mutagenesis, J Mol Biol 335,833-842, 2004).

A fibril structure of Aβ42 with a methionine sulfoxide at position 35proposed by the Riek group is similar to those of Aβ40 fibrils (Luhrs,T., et al., 3D structure of Alzheimer's amyloid-beta(1-42) fibrils, ProcNatl Acad Sci USA 102, 17342-17347, 2005). In an Aβ42 fibril structuremodel, residues D1-L17 are disordered. The residues V18-S26 and I31-A42form in-register parallel β sheets mediated by intermolecular hydrogenbonding and stabilized by salt bridge between D23 and K28. The two βstrands are connected through the loop region residues N27-A30. Electronparamagnetic resonance spectroscopy studies of Aβ40 and Aβ42 fibrilssuggested the presence of a bend-like structure in the residues D23-S26(Torok, M., et al., Structural and dynamic features of Alzheimer's Abetapeptide in amyloid fibrils studied by site-directed spin labeling, JBiol Chem 277, 40810-40815, 2002). Overall, Aβ fibrils display βstrand-loop-β strand structures with the loop formed in the linkerregion.

Recent biophysical and biochemical characterizations have furthersupported the presence of structural rearrangements, particularly in theregion E22-A30 (referred to as the linker region herein), during Aβaggregation from monomers to fibrils via oligomeric intermediates (Lazo,N. D., et al., On the nucleation of amyloid beta-protein monomerfolding, Protein Sci 14, 1581-1596, 2005; Grant, M. A., et al., FamilialAlzheimer's disease mutations alter the stability of the amyloidbeta-protein monomer folding nucleus, Proc Natl Acad Sci USA 104,16522-16527, 2007; Baumketner, A., et al., Amyloid beta-protein monomerstructure: a computational and experimental study, Protein Sci 15,420-428, 2006; Baumketner, A., et al., The structure of the Alzheimeramyloid beta 10-35 peptide probed through replica-exchange moleculardynamics simulations in explicit solvent, J Mol Biol 366, 275-285, 2007;Triguero, L., et al., Molecular dynamics study to investigate the effectof chemical substitutions of methionine 35 on the secondary structure ofthe amyloid beta (Abeta(1-42)) monomer in aqueous solution, J Phys ChemB 112, 2159-2167, 2008; Borreguero, J. M., et al., Folding events in the21-30 region of amyloid beta-protein (Abeta) studied in silico, ProcNatl Acad Sci USA 102, 6015-6020, 2005). Aβ monomer folding to form aturn conformation in the linker region has been postulated to be anintramolecular nucleation event based on the experimental andtheoretical results (Lazo, N. D., et al., On the nucleation of amyloidbeta-protein monomer folding, Protein Sci 14, 1581-1596, 2005; Grant, M.A., et al., Familial Alzheimer's disease mutations alter the stabilityof the amyloid beta-protein monomer folding nucleus, Proc Natl Acad SciUSA 104, 16522-16527, 2007; Baumketner, A., et al., Amyloid beta-proteinmonomer structure: a computational and experimental study, Protein Sci15, 420-428, 2006; Baumketner, A., et al., The structure of theAlzheimer amyloid beta 10-35 peptide probed through replica-exchangemolecular dynamics simulations in explicit solvent, J Mol Biol 366,275-285, 2007; Triguero, L., et al., Molecular dynamics study toinvestigate the effect of chemical substitutions of methionine 35 on thesecondary structure of the amyloid beta (Abeta(1-42)) monomer in aqueoussolution, J Phys Chem B 112, 2159-2167, 2008; Borreguero, J. M., et al.,Folding events in the 21-30 region of amyloid beta-protein (Abeta)studied in silico, Proc Natl Acad Sci USA 102, 6015-6020, 2005). Forexample, the Aβ fragment A21-A30 displayed protease resistance inlimited proteolysis, indicating the formation of a stable structurewithin this sequence⁶ (Lazo, N. D., et al., On the nucleation of amyloidbeta-protein monomer folding, Protein Sci 14, 1581-1596, 2005). Five ofseven familial Alzheimer's disease-linked mutations in Aβ known torender greater aggregation and toxic effects cluster within the part ofthis region, particularly residues E22-D23 (Levy, E., et al., Mutationof the Alzheimer's disease amyloid gene in hereditary cerebralhemorrhage, Dutch type, Science 248, 1124-1126, 1990; Hendriks, L., etal., Presenile dementia and cerebral haemorrhage linked to a mutation atcodon 692 of the beta-amyloid precursor protein gene, Nat Genet 1,218-221, 1992; Kamino, K., et al., Linkage and mutational analysis offamilial Alzheimer disease kindreds for the APP gene region, Am J HumGenet 51, 998-1014, 1992; Nilsberth, C., et al., The ‘Arctic’ APPmutation (E693G) causes Alzheimer's disease by enhanced Abetaprotofibril formation, Nat Neurosci 4, 887-893, 2001; Grabowski, T. J.,et al., Novel amyloid precursor protein mutation in an Iowa family withdementia and severe cerebral amyloid angiopathy. Ann Neurol 49, 697-705,2001). These mutations were found to reduce the stability of thestructure formed by residues A21-A30 and, thereby, possibly promotedstructural arrangements to form high order assemblies (Grant, M. A., etal., Familial Alzheimer's disease mutations alter the stability of theamyloid beta-protein monomer folding nucleus, Proc Natl Acad Sci USA104, 16522-16527, 2007). The presence of a lactam bridge between theside chains of D23 and K28 of Aβ accelerated oligomer and fibrilformation (Sciarretta, K. L., et al., Abeta40-Lactam(D23/K28) models aconformation highly favorable for nucleation of amyloid, Biochemistry44, 6003-6014, 2005). Unlike irregularly structured Aβ monomers insolution, monomeric Aβ bound to affibodies displayed a β hairpinconformation which is largely similar to the β strand-loop-β strandstructure found in fibrils (Hoyer, W., et al., Stabilization of abeta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibitsamyloid formation, Proc Natl Acad Sci USA 105, 5099-5104, 2008; Petkova,A. T., et al., A structural model for Alzheimer's beta-amyloid fibrilsbased on experimental constraints from solid state NMR, Proc Natl AcadSci USA 99, 16742-16747, 2002; Luhrs, T., et al., 3D structure ofAlzheimer's amyloid-beta(1-42) fibrils, Proc Natl Acad Sci USA 102,17342-17347, 2005). The structure of Aβ monomeric β hairpin (=βstrand-turn-β strand), however, differed from that of fibrils in termsof relative orientation of β strands and relevant hydrogen bondingpattern (Hoyer, W., et al., Stabilization of a beta-hairpin in monomericAlzheimer's amyloid-beta peptide inhibits amyloid formation, Proc NatlAcad Sci USA 105, 5099-5104, 2008; Petkova, A. T., et al., A structuralmodel for Alzheimer's beta-amyloid fibrils based on experimentalconstraints from solid state NMR, Proc Natl Acad Sci USA 99,16742-16747, 2002). The structural changes, in particular thearrangement of β strands around the linker region, have beenhypothesized to occur during formation of oligomers and their furtherassociation into fibrils (Hoyer, W., et al., Stabilization of abeta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibitsamyloid formation, Proc Natl Acad Sci USA 105, 5099-5104, 2008). Varyingthe linker sequence and conformation may cause distinct twisting andbending of neighboring β strands, which may be propagated through βstrands running to fibril axes resulting in morphological differences(Fandrich, M., et al., The behaviour of polyamino acids reveals aninverse side chain effect in amyloid structure formation, EMBO J 21,5682-5690, 2002; Bieschke, J., et al., Alzheimer's Abeta peptidescontaining an isostructural backbone mutation afford distinct aggregatemorphologies but analogous cytotoxicity, Evidence for a commonlow-abundance toxic structure(s)? Biochemistry 47, 50-59, 2008;Sciarretta, K. L., et al., Spatial separation of beta-sheet domains ofbeta-amyloid: disruption of each beta-sheet by N-methyl amino acids,Biochemistry 45, 9485-9495, 2006; Makabe, K., et al., Atomic structuresof peptide self-assembly mimics, Proc Natl Acad Sci USA 103,17753-17758, 2006).

Low molecular weight Aβ species, such as monomers and dimers, are nottoxic (Haass, C., et al., Soluble protein oligomers inneurodegeneration: lessons from the Alzheimer's amyloid beta-peptide,Nat Rev Mol Cell Biol 8, 101-112, 2007; Kayed, R., et al., Commonstructure of soluble amyloid oligomers implies common mechanism ofpathogenesis, Science 300, 486-489, 2003; Kayed, R., et al.,Permeabilization of lipid bilayers is a common conformation-dependentactivity of soluble amyloid oligomers in protein misfolding diseases, JBiol Chem 279, 46363-46366, 2004; Klyubin, I., et al., Amyloid betaprotein immunotherapy neutralizes Abeta oligomers that disrupt synapticplasticity in vivo, Nat Med 11, 556-561, 2005). Although the cause ofneurodegeneration in AD is not fully understood, recent studies havesuggested that toxicity should result from the generation of solubleintermediate aggregate species rather than from the formation offibrillar species (Id., Glabe, C. G., Common mechanisms of amyloidoligomer pathogenesis in degenerative disease, Neurobiol Aging 27,570-575, 2006). However, Aβ fibrils could serve as a potential pool oftoxic species through dissociation or dynamic exchanges with otheraggregates (Haass, C., et al., Soluble protein oligomers inneurodegeneration: lessons from the Alzheimer's amyloid beta-peptide,Nat Rev Mol Cell Biol 8, 101-112, 2007; O'Nuallain, B., et al.,Thermodynamics of A beta(1-40) amyloid fibril elongation, Biochemistry44, 12709-12718, 2005; Martins, I. C., et al., Lipids revert inert Abetaamyloid fibrils to neurotoxic protofibrils that affect learning in mice,EMBO J 27, 224-233, 2008). The detailed description of how these toxicspecies lead to generation of AD symptoms can be found elsewhere (Haass,C., et al., Soluble protein oligomers in neurodegeneration: lessons fromthe Alzheimer's amyloid beta-peptide, Nat Rev Mol Cell Biol 8, 101-112,2007; Lansbury, P. T., et al., A century-old debate on proteinaggregation and neurodegeneration enters the clinic, Nature 443,774-779, 2006). These toxic oligomers are kinetic intermediates, and candisplay changes in conformation and toxic effects by subtleenvironmental changes (Teplow, D. B., et al., Elucidating amyloidbeta-protein folding and assembly: A multidisciplinary approach, AccChem Res 39, 635-645, 2006; Wetzel, R., et al., Plasticity of amyloidfibrils, Biochemistry 46, 1-10, 2007; Haass, C., et al., Soluble proteinoligomers in neurodegeneration: lessons from the Alzheimer's amyloidbeta-peptide, Nat Rev Mol Cell Biol 8, 101-112, 2007; Kayed, R., et al.,Common structure of soluble amyloid oligomers implies common mechanismof pathogenesis, Science 300, 486-489, 2003).

The present invention is a peptide probe that generates different levelsof fluorescence signals upon recognition of distinct Aβ assembliesthrough its conformational change (FIG. 1B). Peptide probes contain theN-terminus, HCD and the C-terminus of Aβ, and the ‘signal domain’, whichreplaces the linker region of Aβ. The signal domain is responsible forconformation-dependent fluorescence of a nontoxic, membrane-permeablebiarsenical dye, FlAsH (Adams, S. R., et al., New biarsenical ligandsand tetracysteine motifs for protein labeling in vitro and in vivo:synthesis and biological applications, J Am Chem Soc 124, 6063-6076,2002). A peptide probe displayed an increase in FlAsH fluorescenceintensity when mixed with Aβ oligomers, but not monomers/dimers andfibrils. Unlike antibody-based methods, detection of Aβ oligomers usinga peptide probe could occur rapidly without introducing significantperturbation of Aβ samples by virtue of the functional linkage betweenrecognition and generation of signals. This functional coupling mayfurther enable rapid identification of a peptide probe specific forcertain forms of Aβ, small or large, under a well-defined solutioncondition from a diverse library. Taken together, the peptide probe ofthe present invention holds a promise in rapid and specific detection ofAβ oligomers.

Materials and Methods

Materials

PG46, an illustrative peptide probe of the present invention, wassynthesized through solid-phase chemistry, purified by reverse-phaseHPLC, lyophilized and confirmed by MALDI-TOF mass spectrometry byGenScript (Piscataway, N.J., USA). Lyophilized Aβ40 and Aβ42 werepurchased from Anaspec (San Jose, Calif., USA) or W. M. Keck FoundationBiotechnology Resource Laboratory at Yale University (New Haven, Conn.,USA). An antibody recognizing the N-terminus (D1-K16), 6E10, waspurchased from Covance (Princeton, N.J., USA). An oligomer-specificpolyclonal antibody, A11, was purchased from Invitrogen (Carlsbad,Calif., USA). A precision column pre-packed with Superdex 75 waspurchased from GE Healthcare (Buckinghamshire, England, UK). All otherchemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA) unlessotherwise stated.

Other peptides can be developed for use as peptide probes. Anotherillustrative peptide developed for the present invention that may provesuitable as a peptide probe is PG38. PG38 and other suitable peptidescan be prepared and utilized in a manner similar to that disclosed belowin connection with PG46.

PG46 Sample Preparation

For initial dissolution of PG46, the lyophilized PG46 was solubilizedwith hexafluoroisopropanol (HFIP) at 1 mg peptide/2 ml HFIP for 3 hr.The PG46 in HFIP was then aliquoted into 20 vials (0.05 mg peptideeach). The aliquoted PG46 in HFIP was lyophilized overnight. Thelyophilized PG46 was stored at −80° C. until use. PG46 solutions werefreshly prepared every time by solubilization of the HFIP-treated,re-lyophilized PG46 with dimethyl sulfoxide (DMSO) containing 10 mM2-mercaptoethanol at 5 mg peptide/1 ml DMSO (≈1 mM PG46) for 1 hr. PG46in DMSO was subsequently diluted by 100-fold into aqueous bufferscontaining Aβ (see “Aβ Sample preparation” below), unless otherwisementioned. A similar dilution was made into the same buffers without Aβas a control. The final concentration of PG46 and 2-mercaptoethanol was0.05 mg/ml (≈10 μM) and 100 μM, respectively, unless otherwise stated.

Aβ Sample Preparation.

Aβ samples were prepared according to the established protocols wherelyophilized Aβ was solubilized with DMSO (referred to as “DMSOprotocol”), HFIP (referred to as “HFIP protocol”) and 8M urea/pH 10(referred to as “urea protocol”) prior to dilution into phosphatebuffers containing NaCl (Kayed, R., et al., Common structure of solubleamyloid oligomers implies common mechanism of pathogenesis, Science 300,486-489, 2003; Kayed, R., et al., Permeabilization of lipid bilayers isa common conformation-dependent activity of soluble amyloid oligomers inprotein misfolding diseases. J Biol Chem 279, 46363-46366, 2004; Kayed,R., et al., Fibril specific, conformation dependent antibodies recognizea generic epitope common to amyloid fibrils and fibrillar oligomers thatis absent in prefibrillar oligomers, Mol Neurodegener 2, 18, 2007;Stine, W. B., Jr., et al., In vitro characterization of conditions foramyloid-beta peptide oligomerization and fibrillogenesis, J Biol Chem278, 11612-11622, 2003; Dahlgren, K. N., et al., Oligomeric andfibrillar species of amyloid-beta peptides differentially affectneuronal viability, J Biol Chem 277, 32046-32053, 2002; Kayed, R., etal., Annular protofibrils are a structurally and functionally distincttype of amyloid oligomer, J Biol Chem 284, 4230-4237, 2009; Kim, J. R.,et al., Urea modulation of beta-amyloid fibril growth: experimentalstudies and kinetic models, Protein Sci 13, 2888-2898, 2004; Kim, J. R.,et al., Mechanism of accelerated assembly of beta-amyloid filaments intofibrils by KLVFFK(6), Biophys J 86, 3194-3203, 2004). In the DMSOprotocol, lyophilized Aβ40 was first dissolved in HFIP at ˜4 mgpeptide/1 ml HFIP for overnight at a room temperature. Aβ in HFIP wasthen lyophilized again and stored at −80° C. until use. TheHFIP-treated, re-lyophilized Aβ was resolubilized with 50 μl of DMSO permg of peptide for 20 min, followed by direct dilution into pre-filteredphosphate-buffered saline with azide ((PBSA) 0.01 M Na2HPO4/NaH2PO4,0.15 M NaCl, 0.02% (w/v) NaN3, pH 7.4). In the HFIP protocol, 2.5 mg/mlof Aβ in HFIP was 10-fold diluted into PBSA and the HFIP was evaporatedby applying a gentle stream of N₂ for at least 3 hrs. In the ureaprotocol, 8M urea was first prepared in 10 mM glycine-NaOH buffer, pH10, then filtered through 0.22 μm filters. Lyophilized Aβ was thensolubilized at a concentration of 10-12 mg/ml using prefiltered 8M urea,pH 10 for 30 min. Samples were then diluted into filtered PBSA. In allcases, PBSA was filtered through 0.22-μm filters and samples prepared inglass vials.

Aβ40 and Aβ42 monomers/dimers samples were obtained by injection of Aβin aqueous buffers, freshly prepared according to the DMSO and ureaprotocols, to the size exclusion chromatography (SEC) column(superdex75) on a GE fast protein liquid chromatography (FPLC) systemfollowed by fractionation (FIG. 4). Aβ40 oligomer samples were obtainedby incubation of 1 mg/ml Aβ40 in aqueous buffers, prepared according tothe DMSO protocol, at 37° C. for 3 days without stirring. No significantprecipitation occurred after this incubation (data not shown). Thesamples contained 5% DMSO (v/v). Samples of oligomeric Aβ40 Dutch andAβ40 Artic mutants were similarly obtained by incubation of Aβ40 Dutchand Aβ40 Artic in aqueous buffers, prepared according to the DMSOprotocol, at 37° C. without stirring for 2 days and 2-3 hours,respectively. Aβ40 oligomer samples were alternatively prepared by theurea protocol. In this case, 0.25 mg/ml of Aβ40 in PBSA containing 0.4Murea was incubated at 37° C. with constant shaking at 250 rpm in a NewBrunswick Scientific Innova TM4230 incubator (Edison, N.J., USA). After24 hrs, Aβ40 solutions were centrifuged to remove precipitates andsupernatants were immediately used for characterization. For Aβ42oligomer samples, 0.5 mg/ml of Aβ42 in aqueous buffers was preparedaccording to the DMSO protocol, then incubated at 4° C. for 24 hrswithout stirring followed by centrifugation to remove precipitates asdescribed previously (Dahlgren, K. N., et al., Oligomeric and fibrillarspecies of amyloid-beta peptides differentially affect neuronalviability, J Biol Chem 277, 32046-32053, 2002). Aβ40 and Aβ42 fibrilsamples were obtained following the protocol described previously(Kayed, R., et al., Common structure of soluble amyloid oligomersimplies common mechanism of pathogenesis, Science 300, 486-489, 2003;Kayed, R., et al., Permeabilization of lipid bilayers is a commonconformation-dependent activity of soluble amyloid oligomers in proteinmisfolding diseases, J Biol Chem 279, 46363-46366, 2004; Kayed, R., etal., Fibril specific, conformation dependent antibodies recognize ageneric epitope common to amyloid fibrils and fibrillar oligomers thatis absent in prefibrillar oligomers, Mol Neurodegener 2, 18, 2007;Kayed, R., et al., Annular protofibrils are a structurally andfunctionally distinct type of amyloid oligomer, J Biol Chem 284,4230-4237, 2009). Briefly, Aβ in PBSA prepared according to the HFIPprotocol was incubated for 2-4 weeks at a room temperature withcontinuous stirring by a magnetic stir bar at 300 rpm. Samples werecentrifuged to discard soluble fractions and insoluble pellets washed atleast five times and resuspended with PBSA. The mass concentrations ofsoluble Aβ in all samples were determined by ultraviolet (UV) absorbanceat 280 nm or the Bradford assay (Bradford, M. M., A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding, Anal Biochem 72, 248-254, 1976).

Size Exclusion Chromatography (SEC).

Samples were analyzed and fractionated with SEC using a precision columnprepacked with Superdex 75 (GE healthcare, Buckinghamshire, England, UK)on a GE FPLC system, as described previously (Kim, J. R., et al., Ureamodulation of beta-amyloid fibril growth: experimental studies andkinetic models, Protein Sci 13, 2888-2898, 2004; Kim, J. R., et al.,Mechanism of accelerated assembly of beta-amyloid filaments into fibrilsby KLVFFK(6), Biophys J 86, 3194-3203, 2004). Briefly, the mobile phaseflow rate was set at 0.1 ml/min and elution peaks were detected by UVabsorbance at 280 nm. Mobile phase buffer was matched to buffer used forpreparation of Aβ samples. The column was calibrated using the followingproteins as molecular weight standards: insulin chain B (3500),ubiquitin (8500), ribonuclease A (13,700), and bovine serum albumin(67,000). To determine the distribution between smaller species thatcould be resolved on the column (molecular mass 3-70 kDa), and largerspecies that could not be resolved, samples were injected without thecolumn in place; the percent of non-aggregates (monomers+dimers (M+D))was calculated by dividing the M+D peak area by the peak area withoutthe column in place.

Circular Dichroism Spectroscopy

Secondary structure of Aβ in solutions was determined using circulardichroism (CD), collected using an Aviv 62A DS circular spectrometer(Lakewood, N.J., USA) in the far-UV range with 0.1 cm of path length ofcuvette. Ellipticity of sample containing Aβ at each wavelength wasmeasured without dilution. The spectrum of the background was measuredand then subtracted from the sample spectrum.

Dot Blot

One μg of Aβ were applied to a nitrocellulose membrane, allowed to dryat room temperatures. Membrane blocking, washing, incubation withprimary and secondary antibody, development with chemiluminescence wasperformed according to the manufacture's protocol.

FlAsH Fluorescence Measurements.

Freshly prepared PG46 in DMSO at 5 mg/ml with 10 mM 2-mercaptoethanolwas directly diluted by 100-fold into Aβ solutions or buffers without Aβprior to addition of FlAsH. Note that neither volume nor concentrationof Aβ in solution was nearly changed by the addition of 100× PG46. Asanother control, DMSO containing 10 mM 2-mercaptoethanol was 100-folddiluted into Aβ solutions. As a result, all samples (PG46 only, Aβ only,a mixture of PG46+Aβ) contained an equal amount of DMSO and2-mercaptoethanol, respectively. These samples were incubated for 1 hrat a room temperature. Then, 200 μM of FlAsH-(1,2-ethanedithiol (EDT))₂in DMSO was 125-fold diluted into samples of PG46 only, Aβ only and amixture of PG46+Aβ. The samples were then further incubated for anadditional 1 hr prior to FlAsH fluorescence measurements using a PhotonTechnology QuantaMaster QM-4 spectrofluorometer (Birmingham, N.J., USA).Excitation wavelength was 508 nm and emission was monitored at 520-550nm.

Results

Design of a peptide probe prototype. The desired property of peptideprobes is the ability to modulate fluorescence signals throughassociation with Aβ species, in particular oligomers (FIG. 1B). Peptideprobes contain the N-terminus (D1-K16), HCD (L17-A21) and the C-terminus(I31-V40) of Aβ, and the ‘signal domain’ (FIG. 1A). The Aβ N-terminus isincluded as its presence may reduce the self-assembly of peptide probes(Pike, C. J., et al., Amino-terminal deletions enhance aggregation ofbeta-amyloid peptides in vitro, J Biol Chem 270, 23895-23898, 1995). TheHCD and C-terminal domains of Aβ undergo conformational changes duringAβ self-assembly (Teplow, D. B., et al., Elucidating amyloidbeta-protein folding and assembly: A multidisciplinary approach, AccChem Res 39, 635-645, 2006; Lazo, N. D., et al., On the nucleation ofamyloid beta-protein monomer folding, Protein Sci 14, 1581-1596, 2005;Tjernberg, L. O., et al., Arrest of beta-amyloid fibril formation by apentapeptide ligand, J Biol Chem 271, 8545-8548, 1996; Soto, C., et al.,Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain modelof amyloidosis: implications for Alzheimer's therapy, Nat Med 4,822-826, 1998; Liu, R., et al., Residues 17-20 and 30-35 of beta-amyloidplay critical roles in aggregation, J Neurosci Res 75, 162-171, 2004;Christopeit, T., et al., Mutagenic analysis of the nucleation propensityof oxidized Alzheimer's beta-amyloid peptide, Protein Sci 14, 2125-2131,2005; Chimon, S., et al., Evidence of fibril-like beta-sheet structuresin a neurotoxic amyloid intermediate of Alzheimer's beta-amyloid, NatStruct Mol Biol, 2007; Losic, D., et al., High resolution scanningtunnelling microscopy of the beta-amyloid protein (Abeta1-40) ofAlzheimer's disease suggests a novel mechanism of oligomer assembly, JStruct Biol 155, 104-110, 2006; Mastrangelo, I. A., et al.,High-resolution atomic force microscopy of soluble Abeta42 oligomers, JMol Biol 358, 106-119, 2006; Petkova, A. T., et al., A structural modelfor Alzheimer's beta-amyloid fibrils based on experimental constraintsfrom solid state NMR, Proc Natl Acad Sci USA 99, 16742-16747, 2002;Luhrs, T., et al., 3D structure of Alzheimer's amyloid-beta(1-42)fibrils, Proc Natl Acad Sci USA 102, 17342-17347, 2005). Theseconformational changes are utilized in a peptide probe for itsfunctional coupling between binding to Aβ and fluorescence signaling.The HCD and C-terminal domains will also provide a peptide probe withbinding affinity toward Aβ, as they are critical in Aβ self-assembly(Tjernberg, L. O., et al., Arrest of beta-amyloid fibril formation by apentapeptide ligand, J Biol Chem 271, 8545-8548, 1996; Soto, C., et al.,Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain modelof amyloidosis: implications for Alzheimer's therapy, Nat Med 4,822-826, 1998; Liu, R., et al., Residues 17-20 and 30-35 of beta-amyloidplay critical roles in aggregation, J Neurosci Res 75, 162-171, 2004;Christopeit, T., et al., Mutagenic analysis of the nucleation propensityof oxidized Alzheimer's beta-amyloid peptide, Protein Sci 14, 2125-2131,2005). The signal domain is responsible for conformation-dependentfluorescence generation. A tetracystein motif such as CCXXCC (X: anoncystein amino acid) is included in the signal domain (FIG. 1A-B). Thesignal domain forms the conditional binding site of a nontoxic,membrane-permeable biarsenical fluorescent dye, FlAsH (Adams, S. R., etal., New biarsenical ligands and tetracysteine motifs for proteinlabeling in vitro and in vivo: synthesis and biological applications, JAm Chem Soc 124, 6063-6076, 2002). FlAsH becomes fluorescent (>50,000×)very rapidly, within a few seconds to minutes, upon binding to thetetracystein motif (Id.). The structures of adjacent flanking sequenceswould affect the conformation of the tetracystein motif and, therefore,differentiate FlAsH binding and fluorescence (Id.; Ignatova, Z., et al.,Monitoring protein stability and aggregation in vivo by real-timefluorescent labeling, Proc Natl Acad Sci USA 101, 523-528, 2004; Madani,F., et al., Hairpin structure of a biarsenical-tetracysteine motifdetermined by NMR spectroscopy, J Am Chem Soc 131, 4613-4615, 2009;Martin, B. R., et al., Mammalian cell-based optimization of thebiarsenical-binding tetracysteine motif for improved fluorescence andaffinity, Nat Biotechnol 23, 1308-1314, 2005). Based on these findings,we reasoned that functional coupling between Aβ recognition andfluorescence signaling could be achieved by the conformational change ofa peptide probe, particularly in the signal domain and the neighboringHCD and C-terminal domains, upon binding to Aβ. Structural arrangementsof HCD, the C-terminus and the linker region of Aβ are different inmonomers, oligomers and fibrils (Teplow, D. B., et al., Elucidatingamyloid beta-protein folding and assembly: A multidisciplinary approach,Acc Chem Res 39, 635-645, 2006; Fernandez-Busquets, X., et al., Recentstructural and computational insights into conformational diseases, CurrMed Chem 15, 1336-1349, 2008; Lazo, N. D., et al., On the nucleation ofamyloid beta-protein monomer folding, Protein Sci 14, 1581-1596, 2005;Wetzel, R., et al., Plasticity of amyloid fibrils, Biochemistry 46,1-10, 2007; Zhang, S., et al., The Alzheimer's peptide a beta adopts acollapsed coil structure in water, J Struct Biol 130, 130-141, 2000;Lee, J. P., et al., 1H NMR of A beta amyloid peptide congeners in watersolution. Conformational changes correlate with plaque competence,Biochemistry 34, 5191-5200, 1995; Riek, R., et al., NMR studies inaqueous solution fail to identify significant conformational differencesbetween the monomeric forms of two Alzheimer peptides with widelydifferent plaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox), EurJ Biochem 268, 5930-5936, 2001; Hou, L., et al., Solution NMR studies ofthe A beta(1-40) and A beta(1-42) peptides establish that the Met35oxidation state affects the mechanism of amyloid formation, J Am ChemSoc 126, 1992-2005, 2004; Lansbury, P. T., Jr., Evolution of amyloid:what normal protein folding may tell us about fibrillogenesis anddisease, Proc Natl Acad Sci USA 96, 3342-3344, 1999; Walsh, D. M., etal., Amyloid beta-protein fibrillogenesis. Structure and biologicalactivity of protofibrillar intermediates, J Biol Chem 274, 25945-25952,1999; Huang, T. H., et al., Structural studies of soluble oligomers ofthe Alzheimer beta-amyloid peptide, J Mol Biol 297, 73-87, 2000;Nichols, M. R., et al., Growth of beta-amyloid(1-40) protofibrils bymonomer elongation and lateral association, Characterization of distinctproducts by light scattering and atomic force microscopy, Biochemistry41, 6115-6127, 2002; Barghorn, S., et al., Globular amyloid beta-peptideoligomer—a homogenous and stable neuropathological protein inAlzheimer's disease, J Neurochem 95, 834-847, 2005; Chimon, S., et al.,Capturing intermediate structures of Alzheimer's beta-amyloid,Abeta(1-40), by solid-state NMR spectroscopy, J Am Chem Soc 127,13472-13473, 2005; Chimon, S., et al., Evidence of fibril-likebeta-sheet structures in a neurotoxic amyloid intermediate ofAlzheimer's beta-amyloid, Nat Struct Mol Biol, 2007; Losic, D., et al.,High resolution scanning tunnelling microscopy of the beta-amyloidprotein (Abeta1-40) of Alzheimer's disease suggests a novel mechanism ofoligomer assembly, J Struct Biol 155, 104-110, 2006; Mastrangelo, I. A.,et al., High-resolution atomic force microscopy of soluble Abeta42oligomers, J Mol Biol 358, 106-119, 2006; Hoyer, W., et al.,Stabilization of a beta-hairpin in monomeric Alzheimer's amyloid-betapeptide inhibits amyloid formation, Proc Natl Acad Sci USA 105,5099-5104, 2008; Habicht, G., et al., Directed selection of aconformational antibody domain that prevents mature amyloid fibrilformation by stabilizing Abeta protofibrils, Proc Natl Acad Sci USA 104,19232-19237, 2007; Petkova, A. T., et al., A structural model forAlzheimer's beta-amyloid fibrils based on experimental constraints fromsolid state NMR, Proc Natl Acad Sci USA 99, 16742-16747, 2002; Luhrs,T., et al., 3D structure of Alzheimer's amyloid-beta(1-42) fibrils, ProcNatl Acad Sci USA 102, 17342-17347, 2005; Whittemore, N. A., et al.,Hydrogen-deuterium (H/D) exchange mapping of Abeta 1-40 amyloid fibrilsecondary structure using nuclear magnetic resonance spectroscopy,Biochemistry 44, 4434-4441, 2005; Williams, A. D., et al., Alaninescanning mutagenesis of Abeta(1-40) amyloid fibril stability, J Mol Biol357, 1283-1294, 2006; Williams, A. D., et al., Mapping abeta amyloidfibril secondary structure using scanning proline mutagenesis, J MolBiol 335, 833-842, 2002; Torok, M., et al., Structural and dynamicfeatures of Alzheimer's Abeta peptide in amyloid fibrils studied bysite-directed spin labeling, J Biol Chem 277, 40810-40815, 2002; Grant,M. A., et al., Familial Alzheimer's disease mutations alter thestability of the amyloid beta-protein monomer folding nucleus, Proc NatlAcad Sci USA 104, 16522-16527, 2007; Baumketner, A., et al., Amyloidbeta-protein monomer structure: a computational and experimental study,Protein Sci 15, 420-428, 2006; Baumketner, A., et al., The structure ofthe Alzheimer amyloid beta 10-35 peptide probed through replica-exchangemolecular dynamics simulations in explicit solvent, J Mol Biol 366,275-285, 2007; Triguero, L., et al., Molecular dynamics study toinvestigate the effect of chemical substitutions of methionine 35 on thesecondary structure of the amyloid beta (Abeta(1-42)) monomer in aqueoussolution, J Phys Chem B 112, 2159-2167, 2008; Borreguero, J. M., et al.,Folding events in the 21-30 region of amyloid beta-protein (Abeta)studied in silico, Proc Natl Acad Sci USA 102, 6015-6020, 2005). Forthese reasons, it was hypothesized that the binding of a peptide probeto distinct Aβ species would produce different levels of FlAsHfluorescence. The initial peptide probe, PG46 (FIG. 1A), containsCCPGCC, the most effective tetracystein sequence for FlAsH fluorescence(Adams, S. R., et al., New biarsenical ligands and tetracysteine motifsfor protein labeling in vitro and in vivo: synthesis and biologicalapplications, J Am Chem Soc 124, 6063-6076, 2002; Martin, B. R., et al.,Mammalian cell-based optimization of the biarsenical-bindingtetracysteine motif for improved fluorescence and affinity, NatBiotechnol 23, 1308-1314, 2005). Additional residues (HRW and KTF) wereintroduced on both ends of the tetracystein motif in PG46 to improveFlAsH binding and fluorescence (Martin, B. R., et al., Mammaliancell-based optimization of the biarsenical-binding tetracysteine motiffor improved fluorescence and affinity, Nat Biotechnol 23, 1308-1314,2005).

Preparation and Characterization of PG46 Solution

PG46 contains HCD and the C-terminus of Aβ, and therefore is prone toaggregation. Since FlAsH fluorescence of PG46 may depend on structuresof the signal domain and its flanking sequences, which could also beinfluenced by aggregation states, a well-characterized and reproducibleinitial condition was needed to minimize variation from run to run. Tothis end, the lyophilized PG46 was first solubilized at 0.5 mg/ml withHFIP, known to promote formation of a helical structures of manyamyloidogenic peptides including Aβ (Teplow, D. B., Preparation ofamyloid beta-protein for structural and functional studies, MethodsEnzymol 413, 20-33, 2006). As expected, PG46 also displayed apredominant α helical structure as determined by CD (supporting FIG.5A). CD signals were changed over time during the first few minute ofincubation of PG46 in HFIP, then remained unchanged after then(supporting FIG. 5A), implying no further occurrence of dissolution or αhelical formation of PG46. Based on this finding, PG46 was incubated inHFIP for 3 hrs and then lyophilized for all the experiments.

The fresh PG46 solution was prepared by redissolution of theHFIP-treated, lyophilized PG46 with DMSO containing 10 mM2-mercaptoethanl at 5 mg/ml. Then, PG46 solutions were rapidly dilutedinto PBSA. First, the aggregation state of freshly prepared PG46 inaqueous buffers using SEC was determined. To determine the fraction ofPG46 in aggregated versus non-aggregated (monomers/dimers) form, peakareas were compared for identical samples injected with and without theSEC column in place as described previously and results were summarizedin Table 1 (Kim, J. R., et al., Urea modulation of beta-amyloid fibrilgrowth: experimental studies and kinetic models, Protein Sci 13,2888-2898, 2004; Kim, J. R., et al., Mechanism of accelerated assemblyof beta-amyloid filaments into fibrils by KLVFFK(6), Biophys J 86,3194-3203, 2004). The freshly prepared PG46 was mostly monomeric at≦0.002 mg/ml (=0.4 μM). In contrast, oligomerization of PG46 occurredimmediately after dilution into PBSA at ≧0.01 mg/ml (=2 μM). OligomericPG46 was found to be dominantly present at 0.05 mg/ml (Table 1).Filtration of PG46 solution at 0.05 mg/ml with a 50 kDa cut-off membranewas carried out and provided similar results. Nearly all of PG46 inaqueous buffers at 0.05 mg/ml existed as oligomers of >50 kDa in size.No visible precipitate was observed from PG46 solutions for at least 4hrs. Taken together, the results indicate that the predominant fractionof PG46 at 0.05 mg/ml was soluble oligomers. PG46 oligomers wereSDS-labile; it was dissociated into monomers in a SDS-PAGE (data notshown). PG46 oligomers were recognizable by A11 (supporting FIG. 5B), apolyclonal antibody capable of detecting a common backbone structuresfound in oligomers formed by amyloidogenic peptides/proteins withvarious primary sequences. PG46 at 0.05 mg/ml was used for most FlAsHfluorescence measurements described below.

FlAsH fluorescence of 0.05 mg/ml (=˜10 μM) PG46 was measured with anincreasing concentration of FlAsH; the fluorescence intensity leveledoff at >0.5 μM FlAsH (FIG. 1C). This also indicates that the majority ofPG46 molecules were not accessible to FlAsH, presumably because ofoligomeric nature of PG 46. For FlAsH fluorescence measurements with Aβ,0.05 mg/ml of PG46 and 1.6 μM of FlAsH were used.

Characterization of Aβ40 Samples—Monomers/Dimers, Oligomers and Fibrils.

“Aβ40 monomer/dimer” samples, prepared according to the DMSO and ureaprotocols followed by SEC fractionation, displayed no significantfluorescence when mixed with Thioflavin T (ThT) (FIG. 2A), a fluorescentdye specific for β-sheet structures found in amyloid fibrils (LeVine,H., 3rd., Quantification of beta-sheet amyloid fibril structures withthioflavin T, Methods Enzymol 309, 274-284, 1999). No aggregate wasdetected in transmission electron microscopy (TEM) of these samples(FIG. 2B). Aβ40 monomers/dimers prepared according to the urea protocolwere largely irregularly structured as determined by CD (FIG. 2C). “Aβ42monomer/dimer” samples were prepared according to the DMSO protocols. Nostrong ThT fluorescence was observed from these samples as in Aβ40monomers/dimers (FIG. 2A). TEM images of Aβ42 monomer/dimer samplescollected from SEC showed the presence of a dominant fraction ofnon-aggregated species (FIG. 2B).

“Aβ40 soluble oligomer” samples displayed a slightly increased ThTfluorescence intensity compared to Aβ40 monomer/dimer samples (FIG. 2A).ThT fluorescence signals from these samples were significantly lowcompared to those from fibril samples (FIG. 2A). SEC and membranefiltration analyses confirmed that a predominant fraction (−80%) ofthese samples, prepared according to the DMSO and urea protocols, wasoligomeric (>50 kDa) with the remainder being lower molecular weightspecies such as monomers/dimers (data not shown). These samples appearedpredominantly as worm-like curvilinear particles in a TEM image (FIG.2B). “Aβ42 soluble oligomer” samples were prepared following the DMSOprotocol and found to display low ThT fluorescence compared to fibrils(FIG. 2A). Small prefibrillar particles were detected in a TEM image ofthese samples (FIG. 2B). No further separation was made for both Aβ40and Aβ42 oligomeric samples.

“Aβ40 fibril” and “Aβ42 fibril” samples were separated from solublespecies by centrifugation. These samples exhibited much higher ThTfluorescence intensity compared to other Aβ samples (FIG. 2A). Thepresence of mature fibrils in these samples was confirmed by TEM (FIG.2B).

FlAsH Fluorescence.

FlAsH fluorescence of PG46 at 0.05 mg/ml was measured in the presence ofdifferent Aβ species, such as monomers/dimers, soluble oligomers andfibrils. PG 46 was freshly prepared each time and coincubated with Aβsamples. Incubation with Aβ40 monomers/dimers yielded no significantchange of FlAsH fluorescence of PG46 (FIGS. 2A and G). In contrast,FlAsH fluorescence almost doubled when PG46 was mixed with Aβ40 solubleoligomers (FIGS. 2B and H). Incubation of PG46 with Aβ40 fibrilsresulted in a decrease in FlAsH fluorescence (FIG. 2C). FlAsHfluorescence responses of PG46 were similar when mixed with Aβ samplesprepared according to the different protocol (e.g., DMSO vs. ureaprotocols) (FIGS. 2A and G, and B and H). FlAsH fluorescence intensityof PG46 appeared to increase with an increasing mass concentration of Aβsoluble oligomers from 0 to 0.1 mg/ml followed by a level-off with afurther addition of Aβ (data not shown). FlAsH fluorescence intensity ofPG46 was reduced when mixed with Aβ42 monomers/dimers (FIG. 2D) and Aβ42fibrils (FIG. 2F). A slight increase in FlAsH fluorescence intensity wasobserved when PG46 mixed with Aβ42 oligomers. The FlAsH fluorescencesignals of PG46 were low with Aβ42 oligomers compared to Aβ40 oligomers.FlAsH fluorescence of PG46 was found to increase when mixed witholigomers formed by Aβ40 Artic (E22G) and Dutch (E22Q) mutants (FIG.S3).

Amino Acid Sequences.

The following are sequences illustrative of probes developed for thepresent invention:

PG46: DAEFRHDSGYEVHHQKLVFFA

IIGLMVGGVV PG38: DAEFRHDSGYEVHHQKLVFFA

IIGLMVGG The following are sequences of illustrative betaamyloid (Aβ) probed using the present invention: Aβ40:DAEFRHDSGYEVHHQKLVFFA

IIGLMVGGVV Aβ42: DAEFRHDSGYEVHHQKLVFFA

IIGLMVGGVVIA

Other embodiments of the invention include a peptide probe comprisingPG46, a peptide probe consisting essentially of PG46, and a peptideprobe for the detection of amyloid aggregation comprising PG46. Stillother embodiments of the invention include a peptide probe comprisingPG38, a peptide probe consisting essentially of PG38, and a peptideprobe for the detection of amyloid aggregation comprising PG38.

Another embodiment of the invention is a method for producing a peptideprobe comprising the steps of:

a) lyophilizing a peptide;b) solubilizing the lyophilized peptide of step a) withhexafluoroisopropanol (HFIP) at 1 mg peptide/2 ml HFIP for 3 hr;c) aliquoting the peptide of step b) in HFIP into 20 vials of 0.05 mgpeptide each;d) lyophilizing the aliquoted peptide of step c) in HFIP;e) solubilizing the lyophilized peptide of step d) with dimethylsulfoxide (DMSO) containing 10 mM 2-mercaptoethanol at 5 mg peptide/1 mlDMSO (>>1 mM PG46) for 1 hr; andf) diluting the peptide of step e) in DMSO by 100-fold into aqueousbuffers containing Aβ.

In this method, illustrative peptides include PG46 and PG38.

The foregoing detailed description of the preferred embodiments and theappended figures and references, which are incorporated herein in theirentireties, have been presented only for illustrative and descriptivepurposes. They are not intended to be exhaustive and are not intended tolimit the scope and spirit of the invention. The embodiments wereselected and described to best explain the principles of the inventionand its practical applications. One skilled in the art will recognizethat many variations can be made to the invention disclosed in thisspecification without departing from the scope and spirit of theinvention.

TABLE 1 Population of Soluble Species in PG46 Samples Concentration(mg/ml) 0.002 0.01 0.05 0.05 Method of SEC ^(a)) SEC ^(a)) SEC ^(a))Membrane determination filtration ^(b)) % of monomer + 90 ± 35 20 ± 4 <1 NA dimer ^(c)) % of oligomer ^(d)) ~10 80 ± 4 >99 NA % of species NANA NA ~1 with <50 kDa % of species NA NA NA 20 ± 4 with 50-100 kDa % ofspecies NA NA NA 80 ± 4 with >100 kDa ^(a)) Population of solublespecies determined by the size exclusion chromatography (SEC) usingSuperdex75. The data were analyzed as described previously⁷⁹⁻⁸⁰. ^(b))Population of soluble species determined by filtration with membraneswith cut-off pore sizes of 50 and 100 kDa. The percentage of eachfraction was calculated by measuring the absorbance at 280 nm of samplesbefore and after filtration. ^(c)) Apparent MW <10 kDa as determined bySEC. ^(d)) Apparent MW >70 kDa as determined by SEC. Errors representone standard deviation (n ≧ 3)

1. A method for the rapid and specific detection of amyloid (Aβ)aggregation comprising: a) producing a peptide probe by lyophilizing apeptide, solubilizing the peptide with hexafluoroisopropanol,lyophilizing the peptide; solubilizing the peptide with dimethylsulfoxide, and then diluting the peptide in dimethyl sulfoxide; and b)measuring fluorescence signals generated by the peptide probe, whereby,a presence of fluorescence signals indicates the detection of Aβaggregates.
 2. The method as claimed in claim 1, wherein the peptideprobe generates fluorescence signals rapidly upon recognition of variousAβ aggregates without significant perturbation of samples.
 3. The methodas claimed in claim 1, wherein the peptide probe displays an increase influorescence signals upon coincubation with Aβ oligomers, but not withmonomeric/dimeric species or fibrils.
 4. The method as claimed in claim1, wherein the detection of Aβ aggregates occurs within two hourswithout any additional sample preparation and incubation steps.
 5. Themethod as claimed in claim 1, wherein the peptide probe generatesdifferent levels of fluorescence signals upon recognition of distinct Aβassemblies through its conformational change.
 6. The method as claimedin claim 5, wherein the peptide probe contains the N-terminus, HCD andthe C-terminus of Aβ, and the signal domain, which replaces the linkerregion of Aβ.
 7. The method as claimed in claim 6, wherein the signaldomain is responsible for conformation-dependent fluorescence of anontoxic, membrane-permeable biarsenical dye, FlAsH.
 8. The method asclaimed in claim 7, wherein the peptide probe displays an increase inFlAsH fluorescence intensity when mixed with Aβ oligomers, but not whenmixed with monomers/dimers or fibrils.
 9. The method as claimed in claim1, wherein the peptide probe modulates the fluorescence signals throughassociation with Aβ species.
 10. The method as claimed in claim 9,wherein the Aβ species are oligomers.
 11. The method as claimed in claim10, wherein the peptide probe contains the N-terminus (D1-K16), HCD(L17-A21) and the C-terminus (I31-V40) of Aβ, and the signal domain. 12.The method as claimed in claim 1, wherein the peptide is PG46.
 13. Themethod as claimed in claim 1, wherein the peptide is PG38.
 14. A methodfor the rapid and specific detection of amyloid (Aβ) aggregationcomprising the steps of: a) producing a peptide probe by: i)lyophilizing a peptide; ii) solubilizing the lyophilized peptide of stepa) with hexafluoroisopropanol at 1 mg peptide/2 ml HFIP for 3 hr; iii)aliquoting the solubilized peptide of step b) into 20 vials of 0.05 mgpeptide each; iv) lyophilizing the solubilized peptide of step c) inhexafluoroisopropanol; v) solubilizing the lyophilized peptide of stepd) with dimethyl sulfoxide containing 10 mM 2-mercaptoethanol at 5 mgpeptide/1 ml dimethyl sulfoxide (>>1 mM PG46) for 1 hr; and then vi)diluting the peptide of step e) in dimethyl sulfoxide by 100-fold intoaqueous buffers containing Aβ; and b) measuring fluorescence signalsgenerated by the peptide probe, whereby, a presence of fluorescencesignals indicates the detection of Aβ aggregates.
 15. The method asclaimed in claim 14, wherein the peptide is PG46.
 16. The method asclaimed in claim 14, wherein the peptide is PG38.